Periodontal diseases are oral infections that lead to inflammation of the gingiva, alveolar bone loss, and eventual loss of teeth. There is overwhelming evidence that these infections are caused by specific microorganisms particularly gram negative bacteria. The 1989 consensus report of the Proceedings of the Bacteroides gingivalis were implicated most frequently. A variety of pathogenic mechanisms and virulence factors have been associated with the periopathic potential of these organisms. Currently, however, there is no agreement or even consensus as to the actual mechanisms of pathogenesis or virulence factors among these organisms. Periodontic diseases are characterized by highly acute inflammation and destruction followed by periods of quiescence. It is our hypothesis that Aa invasion of epithelial cells and eventually connective tissues is a virulence factor and plays a role in the characteristic periodicity of periodontitis. The intracellular organisms may serve as reservoirs from which the bacteria recolonize the subgingival root surfaces after periodontal therapy. We recently developed an in vitro cell invasion model that demonstrates Aa invades oral epithelial cells. Smooth isogenic variants of the rough phenotype are most invasive while the rough phenotype is most adhesive. These data suggest there is a coordinated regulation of virulence factors. We are now in a position to understand the factors involved in the molecular biology of adhesion to and invasion of epithelial cells by Aa. The specific aims of the proposed research are to 1) develop the tools for the molecular analysis of Aa adhesion and invasion factors; 2) determine the bacterial factors involved in Aa adhesion to and entry and localization within host cells; 3) determine the intracellular survival and multiplication of internalized Aa, and the mechanisms of release of Aa. 4) Determine the molecular bases for Aa adhesion and invasion by cloning the adhesion and invasion genes; determining the loci involved in the adhesion and invasion phenotype by TnphoA mutagenesis; sequencing the cloned gene(s); and purifying the expressed proteins. 4) Construct invasion and adhesion minus mutants of Aa by allelic recombination of wild-type genes with mutated cloned genes. Our long term goals are to understand the overall nature of the adhesion and invasion processes utilized by Aa and to gain some insight as to the importance of adhesion and invasion in the establishment of periodontal infection.